New quinazolin-2,4-dione derivatives incorporating acylthiourea, pyrazole and/or oxazole moieties as antibacterial agents via DNA gyrase inhibition

This article contributes to the search for new therapeutic agents for treatment of diseases caused by bacterial pathogens. In this study, a new series of compounds incorporating numerous bioactive moieties such as quinazolin-2,4-dione, acylthiourea linkage, and/or five membered nitrogen heterocycles (pyrazole and oxazole) 2–5a–c was described to identify new antibacterial drug candidates via inhibition of DNA gyrase enzyme. The precursor N-[N′-(2-cyano-acetyl)-hydrazinocarbothioyl]-4-(2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-benzamide 2 was prepared by treatment of compound 1 with ammonium thiocyanate and cyanoacetic acid hydrazide through multicomponent reaction (MCR). In addition, compounds 3a–d and 4a–b were synthesized by treatment of 2 with aromatic aldehydes and/or ketones through Knoevenagel reaction, affording high purity products in satisfactory yields. Moreover, new heterocyclic moieties such as pyrazole and/or oxazole attached to quinazolin-2,4-dione core 5a–c were synthesized by treatment of 3c with different nucleophilic reagents like hydrazine, phenyl hydrazine and hydroxyl amine, respectively. Subsequently, the obtained products were structurally characterized by IR, 1H-, 13C-NMR, and MS analyses. The minimum inhibitory concentration (MIC) and antibacterial potency of all compounds were estimated against two G−ve (E. coli and P. aeruginosa), and two G+ve bacteria (B. subtilis and S. aureus). Encouragingly, compound 3c demonstrated the best antibacterial activity against all the strains of the tested pathogenic bacteria at low concentrations compared with the standard drug, Ciprofloxacin. Electron withdrawing groups such as –NO2 and –Cl enhance the antibacterial activity. Next, a molecular docking study between the synthesized derivatives and the target enzyme, DNA gyrase enzyme (PDB: 2xct) was undertaken to investigate intermolecular interactions between the compounds and target enzyme.


Introduction
Infectious diseases caused by bacteria pathogens, are the main cause of public health problems throughout the world. 1 Hence, many types of drugs have been developed and used to treat various types of infections caused by bacteria. 2However, several reports have reported on pathogenic microorganisms that improve resistance to most available drugs. 3 The problem is exacerbated by the rapid development of new pathogenic microorganisms. 4Consequently, the treatment of cancer and infectious diseases continues to be challenging at this time and requires continuous research to develop new, effective, and safe antibiotics.
By searching for antibacterial inhibitors, it was found that quinazolinone derivative I signicantly inhibits the activity of S. aureus DNA gyrase with IC 50 value of 0.25 mM. 5 In addition, acylthiourea derivative II inhibits S. aureus DNA gyrase with IC 50 value of 14.59 mM. 6Whereas, pyrazole derivative has attracted great interest due to its signicant activity against B. subtilis DNA gyrase with IC 50 value of 0.25 mM, 7 as shown in Fig. 1.
On the other hand, molecular hybridization is a rational design strategy for new ligands, based on the recognition of drug-like subunits in the molecular structure of two or more known bioactive derivatives. 8Even today, compounds containing quinazolin-2,4-dione scaffold [9][10][11] represent an endless inspiration for the design and development of new agents showing a wide range of biological properties (Fig. 2).Acylthiourea is a functional group presents in many biologically active agents with antimicrobial, anticancer, and antioxidant [12][13][14] (Fig. 2).][14][15][16] According to the recent studies, quinazolin-2,4-dione fragment attached to acylthiourea core and/or ve membered nitrogen heterocycles possess biological activity, 17 including anticancer, 18 anti-malarial, 19 antibacterial, 20 antiviral, 21 antifungal 22 and anti-inammatory. 23Our targeted molecules structure design has created from marketed available drugs.
Inspired by the above mentioned, we aim to design new series of compounds incorporating various bioactive cores such as quinazolin-2,4-dione, acylthiourea linkage and/or ve membered nitrogen heterocycles 2-5a-c as more effective DNA gyrase enzyme inhibitors, and investigate their antibacterial activities against E. coli and P. aeruginosa of two G−ve bacteria strains, and B. subtilis and S. aureus of two G+ve bacterial strains.In this study, rational approaches such as in silico docking study and ADMET (adsorption, distribution, metabolic, excretion, and toxicity) properties were utilized to select the best compounds to serve as potential antibacterial inhibitors.The synthesis of quinazolin-benzamide derivatives 3a-d was achieved by the reaction of compound 2 with various aromatic aldehydes such as, benzaldehyde, p-chlorobenzaldehyde, pnitrobenzaldehyde and p-hydroxy benzaldehyde, respectively, through Knoevenagel reaction.The success of the formation of the new compounds 3a-d was structurally supported by spectral data.For instance, structure of compound 3a was conrmed by IR spectrum which exhibited absorption bands for NH, CN, CO and CS at n 3219, 2222, 1720 and 1240 cm −1 , respectively.However, 1 H-NMR spectrum of 3a clearly showed the presence of NH protons as singlet signals at d 11.66, 10.77, 10.75 ppm, along with aromatic protons as multiplet signals in the region of d 7.24-8.33ppm.Moreover, 13

Antibacterial activity
The global signicance of antibiotic resistance as a severe danger to public health, resulting in diminished effectiveness of antibiotics, has been well acknowledged.Therefore, the development of novel medication candidates with broad-spectrum antibacterial properties could help address these difficulties.In this study, the antibacterial efficacy of the prepared compounds 2-4b toward various pathogenic microbes was estimated.The MIC was stated, as represented in Table 1.
It was noticed that the tested compounds revealed a considerable wide broad spectrum of antibacterial potency against most of the strains of the tested pathogenic microbes.Meanwhile, 3c exhibited the most remarkable antibacterial efficacy toward all the strains of the tested pathogenic bacteria at lower concentrations than the reference drug, ciprooxacin.

In silico molecular docking studies
In order to investigate the molecular mechanism of the antibacterial action of the target compounds, we performed molecular docking experiments, 24 using the MOE program.The molecular docking tests demonstrated favorable interactions between the synthesized derivatives and the target protein, DNA gyrase enzyme (PDB: 2xct). 25Fig. 4-9 exhibited the 2D and 3D representations of docking styles of target compounds 2, 3a-c and 4a,b with active site of DNA gyrase enzyme.Compound 2 formed two HB with Gly1332 and Gln1267 via carbonyl oxygen and thiourea sulfur atoms, respectively.Additionally, quinazoline ring of compound 2 made pi-H with Asn1269 (Fig. 4).Compound 3a formed dual HB with Lys1276 and Ser1330 via nitrile nitrogen and thiourea sulfur atoms, respectively.Moreover, the hetero ring of quinazoline of compound 3a interacts with adenine DA18 (Fig. 5).Compound 3b formed HB with guanine DG16 through thiourea group and dual pi-H interactions with Gln1267 through quinazoline moiety (Fig. 6).Out of all target compounds, 3c showed intricate interactions with DNA gyrase which aligns with its high antimicrobial activity.Compound 3c formed two HB with Lys1043 and Ile1175 via carbonyl and thiourea groups, respectively (Fig. 7).In addition, the quinazoline ring of compound 3c had dual pi-H interactions with adenine DA18 and another pi-H interaction between distal phenyl ring and Ser1085.Compound 4a formed HB with Gln1267 and two pi-H interactions with Arg1033 and adenine DA18 (Fig. 8).Finally, 2-oxo moiety of quinazolin-2,4dione of compound 4b interact with magnesium MN2492 (Fig. 9).Also, compound 4b formed two HB with Lys460 and adenine DA13.
One of characteristic structural features of target compounds is their ability to form stable intramolecular HB between carbonyl oxygen and hydrogen of thiourea moiety which results in a highly stable classical pseudo six-membered ring.This intramolecular HB was clear in the most stable poses of compounds 2, 3c, 4a and 4b.Along with insertion of unsaturation, intramolecular HB is considered one of the skillful rigidication strategies in drug design.Thanks to the intramolecular HB bond, compound 3c, for example, adopts more extended conformation that enhances its interactions with bonding site of DNA gyrase (Fig. 10).

Physicochemical and pharmacokinetics prediction
In order to reach the clinic, potential drug candidate must exhibit a reasonable pharmacokinetic prole.Consequently, the physicochemical and pharmacokinetic characteristics of the   2-4.All target compounds were predicted not to cause centrally adverse effects as they predicted not to pass blood brain barrier.All target compounds were predicted to be resistant to P-gp efflux.The expected impact of the target drugs on CYP450 enzymes, namely CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, suggests that there is a low likelihood of drugdrug interactions occurring.All target compounds were expected not to be inhibitors for all mentioned CYP enzymes except CYP2C9 and CYP3A4.Compound 2 has a molecular weight of less than 500 without any Lipinski violations.The rest of target compounds have molecular weights 510-661 g mol −1 which is considered the only Lipinski violation of compounds 3a, 3b and 4a.Compounds 3c, 3d, 4b, 5b and 5c have addition Lipinski violation because nitrogen and oxygen more than ten atoms.All target compounds have only one violation in Egan lter.All compounds have no violations in Muegge lter except compounds 5a and 5b.In relation to the Abbott bioavailability score, it was observed that target compounds 2, 3a, and 3b exhibit oral absorption comparable to that of ciprooxacin.

Antibacterial potency
The minimum inhibitory concentrations (MICs) of the prepared derivatives were estimated against E. coli and P. aeruginosa of two G−ve bacteria strains, and B. subtilis and S. aureus of two G+ve bacteria strains.The pathogens under study were provided by Al-Azhar University, Egypt.They were cultivated in Mueller Hinton broth at 35 ± 2 °C for 24 h.The antimicrobial activity and MIC were carried out as described by Qader et al. (2021). 26

In silico molecular docking studies
Molecular docking studies on the synthesized compounds were performed using MOE soware developed by the Chemical Computing Group ULC, Montreal, Canada.Also, the representation 2D style of the interactions between the synthesized compounds and the target protein was performed using MOE soware.

Conclusion
This study involved the design, and synthesis of a novel set of hybrid compounds ( Paper RSC Advances membered nitrogen heterocycles.The objective was to assess their potential as antibacterial agents.The in vitro investigations predominantly demonstrated that compound 3c (with electron withdrawing group -NO 2 ) displayed the highest antibacterial efficacy against all tested harmful bacteria strains at low doses, surpassing the conventional medication Cipro-oxacin.The results were also correlated with the molecular docking investigations, which determined that compound 3c exhibited signicant inhibitory activity against the target protein, DNA gyrase enzyme (PDB: 2xct).Thus, it can serve as drug candidate to develop more potent antibacterial agents due to its high inhibition activity against DNA gyrase enzyme.

2. 1
Scheme 1 outlined the synthetic pathway of derivatives 3ad and 4a-b.Initially, precursor 2 was prepared by treatment of compound 1 with ammonium thiocyanate and cyanoacetic acid

Fig. 3
Fig. 3 GC-MS molecular fragmentation of compound 2 and its structural visualization with mass value.

Table 1
The MIC of the newly prepared compounds a a The standard drug is Ciprooxacin, ND: not determined.

Table 4
Drug likeness parameters of target compounds and ciprofloxacin